1. Field of the Invention
The present invention relates generally to the fields of cellular biochemistry and neurophysiology. Generally, the present invention relates to techniques for optically stimulating genetically designated cells throughout the body. More specifically, it relates, inter alia, to techniques for direct activation of neural circuits by optically stimulating groups of genetically designated neurons.
2. Description of the Related Art
Little is known about the architecture of neuronal circuits controlling brain functions. It is unclear how neuronal cells act in concert to encode and convey information or what roles different types of cells in various regions of the brain play in processing and shaping that information. The ability to address such questions would significantly further the understanding of brain function and will better equip medical researchers to devise treatments for brain-related disorders. Unfortunately, both in vivo and in vitro techniques available currently are insufficient.
One in vivo technique used to provide information about brain function involves stimulating an experimental subject with a sensory stimulus, such as a smell, a sound, an image, etc., and measuring the electrical response in the brain with externally positioned electrodes. Alternatively, instead of measuring the response electrically, the response to the stimulus may be observed using functional magnetic resonance imaging (fMRI).
Although such in vivo techniques are helpful in identifying certain general areas correlated with particular brain functions, these techniques typically do not enable one to identify the cellular elements of specific neuronal pathways.
In contrast, various in vitro techniques permit the identification of individual neurons linked in neuronal pathways present within tissue samples or networks made up of mixed ensembles of neuronal cells. However, they do not help to determine what role, if any, those pathways play in the intact animal. For a summary of various in vitro techniques see, e.g., Crick, “The impact of molecular biology on neuroscience,” Phil. Trans. R. Soc. Lond. B, 354:2021–5 (1999) and Zemelman et al., “Genetic schemes and schemata in neurophysiology,” Current Opinion in Neurobiology, 11:409–14 (2001).
One in vitro technique for identifying neuronal pathways involves a microelectrode search for neurons presynaptic to a given neuron. This technique typically involves impaling a first neuron with a first microelectrode and impaling a second neuron with a second microelectrode. The first microelectrode is then used to electrically stimulate the first neuron. The second microelectrode is used to measure any electrical response from the second neuron resulting from stimulation of the first neuron. The first microelectrode is then switched from the first neuron to a number of other neurons, and this stimulation and response procedure is repeated for each such neuron tested. This technique can be, however, both time and labor intensive, and often results in the incomplete elucidation of neuronal pathways, leads to the stimulation of axons of passage, and causes mechanical disturbance or destruction of the tissue.
Another in vitro method for identifying neuronal pathways is disclosed in Farber et al., “Identification of Presynaptic Neurons by Laser Photostimulation,” Science, 222:1025–7 (1983). This method comprises administering a fluorescent dye to all the cells in a neural tissue sample. A neuron present within the neural tissue sample is impaled with a microelectrode, and a laser microbeam is used to illuminate a stained neuron of interest, causing the illuminated neuron to fire an action potential. A synaptic response to the photostimulated neuron by the impaled neuron is then measured using the microelectrode. The response of the impaled neuron to a number of other stained neurons in the tissue sample is then tested by using the laser microbeam to sequentially illuminate other stained neurons.
Unfortunately, the Farber method is based on the light-induced formation of small pores in the neuronal membrane resulting from the photoexcitation of the fluorescent dye. (The pores act as ion channels, resulting in depolarization of the affected cell and the subsequent firing by the affected cell of an action potential.) As a result, a given cell can be photostimulated only a small number of times before it is killed by phototoxic damage. In addition, the fluorescent dye used in Farber has been found to vary in performance for different cell types and is occasionally species-specific.
Callaway et al. (Proc. Natl. Acad. Sci USA, 90:7661–5 (1993)) discloses another in vitro method for identifying neuronal pathways. The Callaway method involves treating a neural tissue sample with L-glutamic acid (4,5-dimethoxy-2-nitrobenzyl) ester. L-glutamic acid (4,5-dimethoxy-2-nitrobenzyl) ester is a caged or inactive form of the neurotransmitter glutamate and is capable of being converted to glutamate using ultraviolet irradiation. The locations of neurons making functional synaptic connections to a neuron of interest are then revealed by recording electrical activity from the neuron of interest while sequentially irradiating other neurons in the sample.
One shortcoming of the in vitro techniques described above that involve photostimulation (e.g., the Farber technique and the Callaway technique) is that the photostimulation must be confined to a single cell at any given point in time in order to permit an accurate determination of the presynaptic neuron responsible for exciting the electrically-monitored neuron, while in many systems neurons are known to carry information by acting in concert. However, the individual photostimulation of a large number of neurons present in a neural tissue sample can be time and labor intensive and cannot be used to target multiple cells simultaneously.
Another shortcoming of these photostimulation techniques is that they require the use of synthetic indicators or caged compounds, which are difficult to apply to intact tissues or organisms.
Lastly, the brain is typically not homogenous in terms of cell type, but is made up of different types of neurons that are physically arranged in a manner that combines regularity on a large scale with a significant degree of local variability, with neurons of a given cell type typically forming neuronal pathways with other neurons of the same or different cell type. However, such variations in cell type do not always result in easily identifiable visual differences among various cell types. Consequently, although it is often desirable to examine particular cell types in neuronal pathways or networks, it is often impossible to identify these desired cell types for stimulation. This makes application of the above-described photostimulation techniques all the more difficult.
The prior art is deficient in the lack of effective techniques for, inter alia, stimulating groups of neurons or neural circuits in intact tissue or an intact animal system for the purposes of analysis or intervention. The present invention fulfills this long-standing need and desire in the art.